Antibiotic siomycin and a method of producing same



March 19, 1963 HARUO NISHIMURA 3,08 53 ANTIBIOTIC SIOMYCIN AND A METHODOF PRODUCING SAME Filed May 21, 1962 v 2 Sheets-Shoot 1 Absorbcmce 1 l ll l 220 240 1 260 280- 300 my Wave length, millimicrons FIG. -I

INVENIIDR Haruo Nishimura Attorney! ANTIBIOTIC SIOMYCIN AND A METHOD OFPRODUCING SAME Filed May 21, 1962 March 19, 1963 HARUQ NISHIMURA 2Sheets-Sheet 2 m wE 2.226 5 95- 325 m k. w

INVENTOI? Haruo N/Is/rimura Aframm United States Patent The presentapplication is a continuation-in-part of co- 1 pending applicationSerial No. 151,570, filecl November 10, 1961, now abandoned.

This invention relates to a useful antibiotic designated siomycin" and,more particularly, to its production by fermentation, to methods for itsrecovery and conce'ntration from crude solutions, such as fermentationbroths, and to processes for its purification. The invention in-' eludeswithin its scope the antibiotic in dilute forms, as crude concentrates,and in pure crystalline forms. These novel products are especiallyuseful in combatting pathogenie microorganisms.

The new antibiotie is similar to thiostrepton, isolated by Vandeputte etal., with differences in specific rotation, amino acid composition andpaper chromatographic behavior.

The new antibiotic is formed during the cultivation under controlledconditions of a new species of microorganism which has deposited withtheAmerican Type Culture Collection under an accession number ATCC 13989and named Streptomyces sioyaensis n. sp.

This organism was isolated from a sample of soil collectecl from Shioya,Kobe, Japan, andshows the following microbiological characteristics.

Morphological Characteristics The morphological properties of the strainwere obice Physiological Characteristics (1) COLOR CHARACTERISTICSMedium Aerial substratum Soluble mycelium mycelium pigment Syntheticagar White Pale orange-.- Pale orange.

(Ozapek's agar). J Glucose-asparagine .....do. Reddish yellow Paleyellow.

i ar. Calcium malate .-.-.do Yellowisll. Pale yellow orange to orange.

, dark orange. Nutrient egar..... Yellowish Pale yellow to None.

white. gale yellowish rown. Glueose-bouillon.... Whit Dull yellow Do.

- orange. Glucose-peptono .do......... Dark yeliow...- Pale yellow toagar. mic yellowbrown. Potato glucose a ar. do. -....do. None. Solublestarch aaar. do Pale yellow.-... Do. Glycerol-asparaglne White to yell-Reddish yellow D0.

agar. owish to yellow white. orange.

2) BIOCHEMICAL onaaso'rama'rroa The utilization: of w en" and nitrogensource: by an organism are shown in the following table wherein themarks and indicate increasing utilization and. g

in the case of the mark. the source ll not utilized.

served on glucose-asparagin agar and detailed observa- C b n sourc s;Raoul: tions were made by the agar-cylinder method (see Nishi- Glycerolmura ct al., J. Antibiotics, Set. A, 10 (5): p. 227 (1957)). 0 GlucoseThe microscopic observation of the aerial mycelium shows d-Galactosg thelatter to be characterized by forming main stem with Sucrose irregularside branch. Most of the sporophores are Maltose straight to fiexuousand open loops are observed partially. I actnse Cultural CharacteristicsCharacteristics at colony Medium Growth and sporulatlon Surface EdgeHeight Synthetic agar (Czupck's agar) Excellent Powdery, concentricrings... Entire Glucose usparaglno agar do .do Fimbr Pulvlnato toconvex. Calcium malute agar. -.d0 Convex. Nutrient agar ModcratGlucose-bouillon an Excellent Powdery, concentric rln Capitate topulvinate. (llucoscptonc agar d do Umbonate in central. lotnto g ucoscagar... Powdery to velvety Convex to pulvinate. Soluble starch agar GoodPowdery, concentric rings..- Convex.

(Torn starch agar" Nono i Ulyeerol'asparagine agar Excclleut....Powdery, rings Flmbrlcate to laeerate.. Do.

1 Some clear drops appear on the surface of aerial mycelium, but it doesnot become moist and does not torm hygroscopic areas.

= Clear zone is observe Norr..-Growth typo on glucose broth-Ring typetrace sediment, no soluble pigment. Growth res onse to temperature- Goodgrowth at 23 0., poor growtlrat 31 0., no growth at 4s" 0.

dl-Inositol Soluble star Potato starch d-Xylose l-Rhamnose l-Arabinosed-Fructo Corn starch Salicin Nitrogen sources:

dl-Alanine l-Aspartic acid I-Hlsfidine Glycine l-Arginine--- l-Prolinedl-Valin l-Lencine l-Phenylalanine l-Glutamic acid Sodium nitr Ammoniumbiphosphate di-Methionine Ur Sodium nitrite Among the many species ofStreptomyces described in Bergeys Manual of Determinative Bacteriology,Waksman and Lechevaliers Actinomycetes and their Antibi- 35 otics andother literature, Srreptomyces albus appears to be similar to themicroorganism, Streptomyces sioyaensis n. sp. in whitish. aerialmycelium. However, the strain may be differentiated from the species,Streptomyces albus'.

Properties Streptomueu siayssmis Streptomvm aumu Nutrient agar....Reverse, ale yellow to Reverse, bufly brown. gale yel owish brown. SP,brown.

7 P, none. Babouraud's agar.. Reverse, dark yellow Reverse, ochreoustawny orange. SP, pale yelto mars brown.

lowish brown. brown. Soybean iniusion Edge oi colony: flmbri- Edge oicolony: entire agar. cate tolaoerate. AM, AM, white to pale white.Reverse, dark Hayne's gra Reverse 10 yellow orange. SP, pale olive ufl.SP,

dull yellow. none.

Henrieis agar. Edge of colony: tlmbri- Edge oi colony: entire cate.AM,white. Re- AM, white with dull verse, pale yellow. gray. Reverse,blackish brown. Yeast beeiagar... Good growth. Reverse, Slight growth.Reverse, pale yellow. avellaneous.

Distinguishing characteristics, noted in the following table,

are based on actual comparison with two strains of Strepromyces albus,ATCC 3004 and ATCC 3351.

No'rn.-AM, aerial mycclinm; SP, soluble pigment.

As a result of the above observations, the microorganism has beendesignated a new species and named Streptomyces sioyaensis n. sp.

It is to be understood that for the production of siomycin the presentinvention is not limited to the use of Streptomyces sioyaensis n. sp. Itis especially desired and intended to include the use ofsiomycin-producing mutants or variants produced from the describedorganism by various means, such as X-rays, ultra-violet radiation andnitrogen mustards.

In accordance with one aspect of the present invention, the newantibiotic siomycin is produced during cultivation of the microorganism,Streptomyces sioy'aensis n. sp., in an aqueous nutrient medium at atemperature of about 25 to about 32 0., preferably 27 to 29 C., underaerobic conditions. The composition of this nutrient medium may bevaried over a very wide range. Essentially what is required is a carbonsource, a nitrogen source and trace inorganic elements. Examples ofsuitable carbon sources are starch, glucose, glycerol, dextrin, maltose,fructose, sucrose, lactose and molasses. Suitable sources of nitrogenfor the fermentation process include meat extracts, peptone, corn steepliquor, soybean meal, peanut meal, wheat gluten, cotton seed flour, NZamine (enzymatic hydrolyzate of casein) and yeast extracts. Exam-Properties Streptomycu sioyuemio n. sp. flnptomucu albiu (ATCC 3004)Streptomycns albiu (AICC 3351) Morphology oi aerial myoelium... Mainstems iorrn Do not iorm Do not iorm. Buriaoe oi colony Cigar drops andtypical concentric Do not appear Do not appear.

r ngs appear. Growth type on glucose broth..... Ring type Ring-)pelllelet Ring-)pellicla type. Gelatinll ueia' Negative Positive (strongPositive (strong). Corn starc agar 1 N0 grow Thin growth Thin growth.Glucose ssparagiue agar BM reddish llow; 8P, pale yellow. 8M paleyellow; 8P, none SM pale olive; SP, none. Czapek's agar AM, white; M,pale orange AM g ra h white; 8M, grayis AM ogellowish gray; SM, pale w eCalcium malate agar... AM white; BM, yellowish orange; AM, yellowishwhite; SM, yellow- Al, yellowish gray; BM, pale yel- SP, pale yelloworange. ish gra 8P, none. low; Bl, none. Glucose-bouillon agan. AM,white; BM, dull yellow orange AM, yel owish white; BM, pale yel- AM,yellowish gray; 8M, pale yollowish brown. lowish brown. 8M, dark yellow;SP, pale yellow SM, pale yellow; SP, none SM, pale yellow; SP, none.

Glucose-peptone agar...

Note-SM substratum myoelium, AM aerial myoelium, 8P soluble pigment.

The microorganism, Strepromyces sioyaens-is, may also ples of suitablesources of inorganic elements are mineral salts, such as sodiumchloride, potassium chloride, calcium carbonate and potassium phosphate.

Furthermore the existence of trace metal ions such as be differentiatedfrom the thiostrepton-producing strain, Srreptomyces aureus, as shown inthe following table.

Properties Btnplomsea :ioysmis Strepiomym aumu Utilisation oi osr- Poorgrowth: xylose, Good growth: srablnou,

' hamnoee bon sources. No growth: rharnnose, xylose, true arsbinose,n-uotose, tose. Poor growth: sag sslieln. licin. Utalllsatiglnolitso- Nogrowth Good growth.

um tr e Rgd ictgn oi Positive (strong).. Negative.

lB Csapek-Dox agsr.. Edge oi colony: nmbrt- Edge of colony: entire cateto laeerate. AM, brownish white. Beverse. pale yellowish brown.

end of table,

AM, white with smoke ggy medicl blue spots.

verse pale olive but! ith ark giaucous gray spots.

See footnote a side during the fermentation and, in such case, an acidicsubstance such as acetic acid and ammonium sulfate may be added.Generally speaking, the pH may be kept between 5.9 and 8.2, preferablyaround 7. If excessive lo foaming is encountered during thefermentation, antifoaming agents such as vegetable oils, lard oil andpolypropyleneglycoi may be added to the fermentation medium prior to orin the course of the fermentation. The maximum yields of the antibioticsiomycin can be obtained within about 72 to about 120 hours of.fermentation under optimum conditions of temperature and aeration.

After growth of the microorganism, the mycelium may be removed from thefermentation broth by using standard equipment, such as filter-pressesand centrifuges, and thenthe antibiotic siomycin may be recovered fromthe filtrate by a solvent extraction procedure. As, the antibioticsiomycin is retained by the mycelium in appreciable quantities, asolvent extraction procedure is preferably used to recover theantibiotic from the mycelium or the whole broth without the removal ofthe mycelium. Suitable extraction solvents include dioxane, chloroform,N,N-dimethylacetamide and glacial acetic acid. For the extraction of theantibiotic "from larger volumes of broth,

however, an adsorption procedure is superior to an ordinary solventextraction procedure. For instance, the whole broth may be filteredafter the addition of an adeorbent, such as Hyfio Super-Cel(diatomaceous earth), and the resulting cake of adsorbent and myceliummay be eluted with a suitable organic solvent, such as chloroform anddioxane, to extract the antibiotic. The extract may be concentrated anda suitable organic solvent such as .petroleum ether and hexane added toprecipitate th crude active component.

The thus-obtained crude active component is further purified by suitableoperations, such as recrystallisation, chromatography and the like.Examples of suitable recrystallization solvents are dioxane,chloroforrn,methanol, ethanol, butanol, etc. The preferredchromatographic'adsorbents are alumina, silica gel, silicic acid and thelike.

Theiantibiotic siomycin is a yellowish-white or white crystalline solidwhich darkens at about 200 C. and melts with decomposition at about 255to 260' C. It is soluble in dioxane, chloroform, N,N-dimethy1acetamideand glacial acetic acid, slightly soluble in methanol, ethanol, butanoland propylene-glycol and insoluble in ether, acetone, benzene, hexaneand petroleum ether.

Elementary analysis for siomycin is as follows. Found: C 49.26%, H5.40%, N 14.61%. S 8.35%; O 22.38% (by difference). The molecular weightof siomycin is 1650 to 1850 by the Barger-Akiya method (see Akiya etaL,J. Pharm, Soc. Japan, 57, p. 967 (1937)), and 1500 to 1750 by theNiederl method (see Niederl et al., Science, 100, p. 228 (1944)). Theabove analyses correspond to the molecular formula C H N SQ forsiomycin. The specificrotation of siomycin is [04 -90.9 -t2 (c.=1.0l7%in dioxane). 'Ihe ultraviolet absorption spectrum in methanol presentsno maxima (shown in the accompanying drawings, FIG. 1). The infraredabsorption spectrum in a Nujol mull shows the following frequencies:3.05, 5.75, 6.02, 6.12, 6.31, 6.53, 6.60, 6.74, 7.07, 7.24, 7.3l,-7.41,7.58, 7.73, 7.83, 8.09, 8.29, 8.54, 8.67, 8.80, 8.96, 9.12, 9.44, 9.67,9.77, 10.19, 10.35, 10.51, 10.71, 10.97, 11.27, 11.72, 12.36, 12.85,12.93, 13.14, 13.51 and l3.64 (shown in the accompanying drawings, FIG.2).

It gives negative Molisch and positive biuret and Fehling tests. Theacid hydrolysategives a strong ninhydrin test.

Paper chromatographic analysis of siomycin hydrolyzed with 6 Nhydrochloric acid for 24 hours at 100 C. reveals the presence of anumber of ninhydrin positive components, and these components areidentified as follows: valine, alanine, threonine (or cysteine); noother conventional amino acids are found. Thus, the amino acidcomponents existing in the molecule of siomycin are the said four aminoacids. I

Papergram bioautograph of siomycin in a mixture of methanol-aceticacidwvater (25 :3:72) shows an Rf value of 0.12 to 0.14.

Properties Siomycin Thiostrepton Melting point Darkens at about 200C.Darkens at about 200C.

(decom). and melts with deand melts with doeomposltlon at aboutcomposition at about 255 to 200C. 240 to 255C. Elementary O, 40.26; H,5.40; N, C, 49.56; 11, 5.01; N,

analysis. 14.01; .35. 14.63; S, 8.38. Specific rotation... [a]n'90.9(+2)[a],."-60.2(+4) (CL-1.017% in diox- (C.0.548% in dioxan ane).

Amino acids oom- Valine, alanine, threo- Leuelne, alanine,thrcoposition. {11:18, cystlne or (cysnine, cystine.

e no

Rf value for a mix- 0.12 to 0.14 (single spot) 0.38 to 0.40 (singlespot).

ture of methnnolaoetie acid- I water (25:3:72).

Siomycin shows activity against a variety of microorganisms and thefollowing table illustrates the antibiotic spectrum of siomycin,compared withthat of thiostrepton:

Minimum inhibitory concentration, mcg.

per ml. Test organisms Siomycin Thiostrepton 1. Staphylococcus oureus,209 P 0.05 0. 1 2. Bacillus sublills, POI-219 0.05 0.1 3. Sarllna lutea0. ()5 0.05 4. Bacillus nnlhrccls 0.2 0.2 5. Diplococcus pneumoniae,type I. 0. 005 0.01 6. Diplocorcus pnelimoniae, V-type 0.005 0.01 7.0.005 0.01 8. 0. (1)5 0. 005 s. 0. 01 r. 02 10. 0.01 0. 02 ll. 0. 005 0.01 12. 0. 005 0.01 13.

2. 0 r 5.0 14. 2.0 2.0 15. 2.0 2. 0 10. 20. 0 20.0 17. 10.0 200 18. 20020. 0 10. Escherichia coli Umezuwa. 20. 0 20. 0 20. Salmonella typilosa200 200 21. Salmonella paratyphia A 200 20.0 22. Shiqellu dusentmar 2002 0 23. Shigella paradysenteriae, Ohar 200 200 Culture medium: 1 to 4and 19 to 23, beef extract; 5 to 12, beet extract plus 10% rabbit blood;13, Kirchner plus 10% human plas a; 14 to 18,

a heel extract plus glycerol.

Method of testing: 1 to" 12 and 14 to 23, agar-streak dilution method;13,

subsurface culture.

End point observed: 1 to 12 and 10 to 23, no growth after 24 hrs. at 370.; 13, no rowth after 3 weeks at 37 0.; 14 to 17, no growth after 48hrs, at 37 18 no growth after 72 hrs. at 27 0.

From the preceding table, it is seen that siomycin is highly activeagainst grampositive bacteria and mycobacteria, with little or noactivity against gram-negative bacteria or Candida species. Theantibacterial spectrum of siomycin is quite identical with that ofthiostrepton. However, a quantitative difference is observed; asdetermined with the sensitive microorganisms, siomycin possessesapproximately twice the activity-of thiostrepton.

Because of its activity in vitro against gram-positive bacteria achemotherapeutic test in mice with experimental Diplococcus pneumoniaeinfection was set up. Groups of 10 mice weighing 16-18 grams wereinfected by intraperitoncai injection of MLD (minimum lethal dosis) ofDiplococcus pneumoniae, type I. The mice were treated 3, 24, 48, 72 and96 hours after infection, and survival of infected mice was recorded upto 10 days after infection, at which time the tests were terminated.

An aqueous solution of siomycin was prepared by the following method:the antibiotic is dissolved in N,N-dimethylacetamide and diluted to thedesired concentrations with sterile distlled water. The results of thetests are summarized in the following table.

Thus, siomycin is effective by the intravenous route at a dose of 1 mg.per kg. at which 100 percent protection is obtained for the infectedanimals. By subcutaneous administration, 100 percent protection isobtained with a dose of 2 mg. per kg. of body weight.

Acute toxicity studies on siomycin were carried out in mice Weighing 15to 16 g. following a ten day observation period. The intravenous acuteLD for mice was about ,100 mg. per kg. of body weight.

The mice are here standard experimental animals'and the obtained resultsare statistically significant.

The new antibiotic siomycin is useful as an agent for inhibiting thegrowth of gram-positive pathogenic microorganisms. It is useful forsterilizing equipment, for example surgical instruments. It is alsouseful in obtaining pure cultures of single organisms wherea-susceptible organism may be separated from a resistant one.

The following examples are given solely for the purpose of illustrationand are not to be construed as limitations of this invention, manyvariations of which are possible.

Example 1 A nutrient medium (20 litres) is prepared from the Water isadded to the desired volume (20 litres),

After adjusting the mixture to a pH of 7, steam is passed through themixture to sterilize it. Then the nutrient medium is inoculated withStreptomyces sioyaensis n. sp. and cultivated under aeration for aperiod of 72 to 120 hours at 27' C., with shaking until the siomycinconcentration is 40 micrograms perimillilitre. The antiobioticconcentration in the fermentation broth is determined by the paper-discor cup method (see Edwin et al., J. Bacteriology, 50: p. 459 (1945);Nishimura et al., Annual Report of Shionogi Research Laboratory, 11, p.145 (1961)), using Staphylococcus aureus, Terashima or Bacillussubrilis, PCI 219.

One percent by weight of Hyflo Super-Ce! (diatomaceous earth) is addedto the fermentation broth containing the myceliu-m and the mixture isadjusted to a pH of 5.0 with hydrochloric acid. After 30 minutes ofstirring, the mixture is filtered with suction. The filtered HyfloSuper-Cel and mycelial cake is washed with water, and the washed cake iseluted thrice with chloroform with vigorous agitating, by using eachtime a volume of the solvent corresponding to about one-tenth of thevolume of broth. The chloroform extracts are combined and concentratedin vacuo to a small volume. A crude preparation is obtained byprecipitating the concentrated chloroform extract with five volumes ofpetroleum ether or hexane. The precipitate is washed with petroleumether. On drying this precipitate in a vacuum desiccator, a yellowishbrown powder of siomycin (1500 milligrams) is obtained. The dried powderof siomycin is then dissolved in a large amount of chloroform andfiltered. The filtrate is concentrated in vacuo to a small volume. Thecondensate is then poured into a glass column containing chromatographicalumina through which methanol has been previously percolated. Thecolumn is then developed with chloroform. The active chloroformfractions are combined and concentrated in vacuo to a small volume. Bythe addition of methanol to the concentrated chloroform fraction,siomycin is precipitated as crystals. The crystalline precipitate (150milligrams) is recrystallized by dissolving it in dioxane and thenadding methanol to the resulting solution. By this method, white orslight yellowish white crystals of siomycin (120 milligrams) areobtained.

Example 2 A seed medium (150millilitres) is prepared from the followingmaterials:

Grams per litre Glucose 10 NZ amine (enzymatic hydrolysate of caseinmanufactured by Sheflield Chemical Co.) 2 Yeast extract--- 1 Meat extr 1No pH adjusted.

After sterilizing for 15 minutes at 121 C., the medium is inoculatedwith Streptomyces sioyaensis n. sp., shaken for 47 hours at 29 C. andused as inoculum.

A nutrient medium (50 litres) is prepared from the following materialsz-36. Soybean meal Potato star h Calcium carbonate do 2.5 Acetic acidmillilitre 0,2

After potato starch is pre-cooked by heating with acetic acid and water(20 litres) for 15 minutes at 70 C., there are added soybean meal andcalcium carbonate. The whole is made to the desired volume (50 litres)with water. sterilizing for 20 minutes at 120 C., the

Niax Diol PPG-2025 (polypropyleneglycol of average molecular weight 2000and average hydroxyl number 56 manufactured by Union .Carbide ChemicalsCo.) millilitres) is added as an antifoaming agent. Then, the nutrientmedium is inoculated with the above-prepared inoculum,(150 millilitres)and cultivated under aeration for a period of 95 to 98 hours at atemperature of 28 to 29 C. with agitation. During the cultivation, thepH is kept between 7.0 and 7.2, when variations are encountered, by theoccasional addition of acetic acid and the aeration speed is controlledat a half volume of the medium per minute for the initial 16 hours andthen at the one-fifth volume of the medium per minute for the remainderof the period. The antibiotic concentration in the fermentation brothreaches around 100 micrograms per millilitre.

Example 3 Nutrient mediums are prepared as follows:

No pH adjusted.

After potato starch is pre-cooked by heating with acetic acid and water(30 litres) for 15 minutes at C., 7 there are added soybean meal,calcium carbonate and nutrient medium is distributed in a litres-tankand 9 Niax Diol PPG- 2025. The whole is made to the desired volume (50litres) with water.

No pH' adjusted.

After potato starch is pre-cooked by heating with acetic acid and water(200 litres) for minutes at 70 0., there are added soybean meal, calciumcarbonate and Niax Diol PPG-2025. The whole is made to the desiredvolume (550 litres) with water.

Medium A is distributed in a 100 litres seed tank and sterilized bypassing through steam at 121 C. for minutes. The seed medium isinoculated with the inoculum (150 millilitres) prepared as in Example 2and cultivated under aeration (10 litres per minute) for 28 hours at 29C. while agitating (380 rotations per minute).

Medium B is distributedin a 1000 litres'fermenter tank and sterilized bypassing through steam at 121' C. for 20 minutes. To the fermentationmedium, there is added the above-prepared seed broth. The cultivation isperformed under aeration (75 litres per minute) for 84.5 hours at 29 C.while agitating (240 rotations per minute). During the cultivation, thepH is kept at 7.0 to 7.2 by the occasional addition of acetic acid. Theantibiotic concentration in the fermentation broth reaches 41 microgramsper millilitre.

The resultant fermentation broth (500 litres) is treated as in Example 1to yield pure crystals of siomycin (4.6 grams).

Modifications may be made in carrying out the present invention withoutdeparting from the spirit and scope thereof, and I am to be limited onlyby the appended claims.

I claim:

1. A process for producing a new antibiotic, siomycin, which comprisescultivating a strain of Streptomyces sioyaensis in an aqueous nutrientmedium under aerobic conditions, and recovering the accumulatedantibiotic the resulting cake of the 10 merged aerobic conditions andrecovering siomycin from the fermentation broth.

4. A process for producing a new antibiotic, siomycin, which comprisescultivating a strain of Srrepromyces sioyaensis in an aqueous nutrientmedium under submcr-bed aerobic conditions, filtering the ferementationbroth and extracting the filtrate broth with a waterimmiscible organicsolvent.

5. A process for producing a new antibiotic, siomycin, which comprisescultivating a strain of Streptomyces sioyaensis in an aqueous nutrientmedium under submerged aerobic conditions, and extracting thefermentation broth with a water-immiscible organic solvent.

6. A process for producing a new antibiotic, siomycin, which comprisescultivating a strain of streplomyces .rioyaensis in an aqueous nutrientmedium under sub-' merged aerobic conditions, adding an adsorbent to thefermentation broth, filtering the mixture and extracting adsorbent andthe mycelium with awater-immiscible organic solvent.

7. A process for producing a new antibiotic, siomycin,

which comprises cultivating a strain of Streptomyces sioyaensis in anaqueous nutrient medium under submerged aerobic conditions, treating thefermentation broth with an adsorbent at a pH of about 5.0, filtering themixture, extracting the resulting cake of the adsorbent and the myceliumwith a water-immiscible organic solvent, concentrating the extract toobtain a crude product, precipitating crude siomycin from the crudeproduct with addition of an insoluble organic solvent, andchromatographing a solution of the crude siomycin in a soluble organicsolvent with subsequent elution to isolate siomycin.

8. The new antibiotic, siomycin, effective in inhibiting the growth ofgram-positive microorganisms, said antibiotic being a crystal darkeningat about 200 C., and decomposing at about 255 to 260 (3., containing theelements carbon, hydrogen, nitrogen, sulfur and oxygen in substantiallythe following proportions by weight:

Percent Carbon 49.26 Hydrogen 5.40 Nitrogen 14.61 Sulfur 8.35 Oxygen (bydifference) 22.38

References Cited in the file of this patent Vandeputte et al.:Antibiotics Annual, 1955-1956, p. 560.

8. THE NEW ANTIBIOTIC, SIOMYCIN, EFFECTIVE IN INHIBITING THE GROTH OFGRAM-POSITIVE MICROORGANISMS, SAID ANTIBIOTIC BEING A CRYSTAL DARKENINGAT ABOUT 200*C., AND DECOMPOSING AT ABOUT 255 TO 260* C., CONTAINING THEELEMENTS CARBON, HYDROGEN, NITROGEN SULFUR AND OXYGEN IN SUBSTANTIALLYTHE FOLLOWING PROPORTIONS BY WEIGHT: